EP2649452B1 - Method and kit for the in vitro diagnosis of prostate cancer - Google Patents

Description

The present invention relates to a method and a kit for the in vitro diagnosis of prostate cancer.

Prostate cancer is the most common type of cancer in men. Prostate cancer has the characteristic of evolving very slowly. Nevertheless, despite its moderate biological aggressiveness, compared to other types of cancer, it is the second most fatal cancer in men, after lung cancer, tied with colorectal cancer (1). The severity of the cancer depends on the extent of the tumor (local, with nearby or distant metastases) and the type of cancer cells, that is, the degree of malignancy. There are curative solutions, especially for early stages of cancer, and limited locally such as radiotherapy and / or surgery. It is therefore crucial to have a test that allows the earliest possible and reliable diagnosis of prostate cancer.

Prostate cancer can be tracked by the finding of increased concentration of a protein in the blood, prostate specific antigen or PSA. PSA is produced by the prostate and is therefore a specific marker of the organ but it is not specific to the tumor. This is why a high result in a PSA quantification test does not necessarily mean that there is cancer. In fact, an amount of more than four nanograms per milliliter of this protein in the blood is associated with prostate cancer in 25% of cases and another prostate disorder in 75% of cases, especially when benign hypertrophy (BPH), inflammation or infection of the prostate. In addition, although it is relatively sensitive for initial diagnoses and for patient follow-up, this test does not detect all cases of prostate cancer.

More recently, another marker has been reported to be significantly associated with progression and various stages of prostate and prostate cancer. This is Annexin 3 (ANXA3) which belongs to the Annexines family. The The proteins of the Annexin family have the common characteristic of being able to bind to phospholipid membranes in the presence of calcium. To do this, the members of this protein family share structural similarities. Thus, each Annexin contains in its protein sequence domains called "annexin repeat", which are 4 or 8 (4 for Annexin A3), and have a length of about 70 amino acids (2). Each repetitive domain, also referred to as the endonexin domain, is folded into 5 alpha helices and contains a characteristic motif of type 2 (GxGT- [38 residues] -D / E) which makes it possible to fix Ca2 + ions. Annexins of the animal kingdom have variable N-terminal domains, which often carry the functional specificities of proteins. The crystallographic analyzes show that the secondary and tertiary structures of annexins are conserved even if the identity of the amino acid sequences does not exceed 45 to 55%. The 4 repeat domains form a disk-like structure with a slightly convex surface on which the Ca ²⁺ binding sites are located and a concave surface where the N- and C-terminal ends of the protein are found. proximity.

Annexin A3 is a rare annexin, which is expressed only slightly or not at all by the majority of cell types. Conversely, it is very abundant in human neutrophils, where it accounts for about 1% of cytosolic proteins (3, 4). Initially, two isoforms of Annexin A3 have been described, one of 33 kDa of apparent molecular weight, predominantly present in neutrophils, the other of 36 kDa apparent molecular weight, found in monocytes. The difference in molecular weight between the two isoforms does not result from a post-translational modification such as phosphorylation or glycosylation. Very recently, in 2010, it was shown that an alternative splicing phenomenon could be a mechanism for explaining the presence of these two isoforms. Thus, the authors identified two DNA variants complementary to Annexin A3: the first of 973 base pairs (bp) length encodes an entire protein of 323 amino acids corresponding to the 36 kDa isoform; the second with a length of 885 bp does not contain exon III following alternative splicing. When this exon is not present, the open reading frame is interrupted, and the translation becomes possible only from the ATG codon present in exon IV. As a result, the first 39 amino acids of the protein are not expressed, resulting in an N-terminal truncated protein of 284 amino acids, corresponding to the 33 kDa isoform (5).

In addition, Western blot analysis after 2-dimensional electrophoresis (2DE-WB) shows that the 36 kDa isoform migrates as 4 to 6 spots, having different isoelectric points. The 36 kDa isoform identified by a 1-dimensional Western blot corresponds to a group of heterogeneous isoforms (5). It is the 36 kDa Annexine A3 whose level of expression decreases in case of prostate cancer (5-7).

Although Annexin A3 is recognized as a marker for prostate cancer, the methods for detecting it described so far are lacking in specificity because the antibodies used cross-react with other annexins ( WO 2006/125580 ), or lack sensitivity to detect it in a complex environment such as urine ( WO 2007/141043 ). In general, the prior methods lack robustness.

Surprisingly, the inventors have found that, to overcome the aforementioned drawbacks, it was necessary to use a pair of antibodies, one of which is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO. : 1 (amino acids 27-87 of Annexin A3, according to the numbering of the UniProtKB database of the complete ANXA3 Human Accession No. P12429) and the other is directed against the fourth repeated domain of the Native human Annexin A3 whose sequence is identified in SEQ ID NO: 2 (amino acids 258-318, according to the numbering of the UniProtKB database of the complete human ANXA3 Accession No. P12429).

In the description which follows, the sequence of the first repeated domain of native human Annexin A3 is identified in SEQ ID NO: 1, the sequence of the fourth repeated domain of Annexin A3. native human is identified in SEQ ID NO: 2, the sequence of the second repeating domain of native human Annexin A3 is identified in SEQ ID NO: 3, the sequence of the third repeating domain of native human Annexin A3 is identified in SEQ ID NO: 4. The sequence identified in SEQ ID NO: 5 corresponds to the amino acid sequence of complete native human Annexin A3 (UniProtKB Accession No. P12429).

The subject of the present invention is therefore a method for the in vitro diagnosis of prostate cancer according to which a urine sample to be analyzed is brought into contact with two antibodies, a capture antibody and a detection antibody, a of the two antibodies is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the other of the two antibodies is directed against the fourth replicated domain of native human Annexin A3 whose the sequence is identified in SEQ ID NO: 2.

• SNAQRQLIVKEYQAAYG (SEQ ID NO : 10),
• LIVKEYQAAYG (SEQ ID NO : 11)
• IVKEYQAAYGKE (SEQ ID NO : 12),
• KEYQAAYG (SEQ ID NO : 13),
• DLSGHFEHL (SEQ ID NO : 14),
• LSGHFEH (SEQ ID NO : 15),
• et
• KEYQAAYGKELKDDLKG (SEQ ID NO : 22) à la condition que la séquence en acides aminés SEQ ID NO : 22 soit fusionnée du côté N-terminal à une séquence d'au moins 30 acides aminés.

In particular, the antibody directed against the first repeated domain of native human Annexin A3 is chosen from the antibodies directed against an epitope whose amino acid sequence comprises at least 7 consecutive amino acids, preferably at least 8 or at least 9 amino acids or at least 12 consecutive amino acids and not more than 17 consecutive amino acids of SEQ ID NO: 1, and among these there may be mentioned, preferably, the antibodies which are directed against a polypeptide included in SEQ ID NO: 1 whose amino acid sequence is selected from the following sequences:

• SNAQRQLIVKEYQAAYG (SEQ ID NO: 10),
• LIVKEYQAAYG (SEQ ID NO: 11)
• IVKEYQAAYGKE (SEQ ID NO: 12),
• KEYQAAYG (SEQ ID NO: 13),
• DLSGHFEHL (SEQ ID NO: 14),
• LSGHFEH (SEQ ID NO: 15),
• and
• KEYQAAYGKELKDDLKG (SEQ ID NO: 22) provided that the amino acid sequence SEQ ID NO: 22 is fused on the N-terminal side to a sequence of at least 30 amino acids.

Among the antibodies which are directed against the fourth repeating domain of native human Annexin A3, there may be mentioned antibodies which are directed against an epitope whose amino acid sequence whose amino acid sequence comprises at least 7 consecutive amino acids and not more than 50, preferably no more than 45 consecutive amino acids of SEQ ID NO: 2.

• est inclus dans une séquence en acides aminés correspondant à la séquence en acides aminés commençant au résidu 3 et se terminant au résidu 49 de SEQ ID NO : 2,
• comprend en position 6 de SEQ ID NO : 2 un résidu Lys (K),
• comprend en position 6 de SEQ ID NO : 2 un résidu Lys (K) et en position 49 de SEQ ID NO : 2 un résidu Asp (D),
• comprend en position 7 de SEQ ID NO : 2 un résidu Gly (G), en position 8 de SEQ ID NO : 2 un résidu Ile (I) et en position 9 de SES ID NO : 2 un résidu Gly (G),
• comprend en position 3 de SEQ ID NO : 2 un résidu Arg (R), en position 6 des SEQ ID NO : 2 un résidu Lys (K), en position 7 de SEQ ID NO : 2 un résidu Gly (G), en position 8 de SEQ ID NO : 2 un résidu Ile (I), en position 9 de SEQ ID NO : 2 un résidu Gly (G) et en position 49 de SEQ ID NO : 2 un résidu Asp (D).

In particular, the antibodies which are directed against the fourth repeating domain of native human Annexin A3 are selected from the antibodies which are directed against an epitope which:

• is included in an amino acid sequence corresponding to the amino acid sequence beginning at residue 3 and ending at residue 49 of SEQ ID NO: 2,
• comprises at position 6 of SEQ ID NO: 2 a residue Lys (K),
• comprises at position 6 of SEQ ID NO: 2 a Lys (K) residue and at position 49 of SEQ ID NO: 2 an Asp residue (D),
• comprises at position 7 of SEQ ID NO: 2 a Gly residue (G) at position 8 of SEQ ID NO: 2 an Ile residue (I) and at position 9 of SES ID NO: 2 a Gly residue (G),
• comprises at position 3 of SEQ ID NO: 2 an Arg (R) residue at position 6 of SEQ ID NO: 2 a Lys (K) residue at position 7 of SEQ ID NO: 2 a Gly (G) residue, in position 8 of SEQ ID NO: 2 an Ile (I) residue, in position 9 of SEQ ID NO: 2 a Gly (G) residue and in position 49 of SEQ ID NO: 2 an Asp (D) residue.

The positions 6, 49, 7, 8 and 9 determined with respect to SEQ ID NO: 2 correspond respectively to the positions 263, 306, 264, 265 and 266 of the human Annexin A3 identified in SEQ ID NO: 5 which corresponds to the amino acid sequence of complete native Human Annexin A3 (UniProtKB Accession No. P12429).

Preferably, the antibody directed against the first repeated domain of native human Annexin A3, whose sequence is identified in SEQ ID NO: 1, is the capture antibody and the antibody directed against the fourth repetitive domain of the Native human Annexin A3, whose sequence is identified in SEQ ID NO: 2, is the detection antibody.

The antibodies specific for the first repeated domain of human native Annexin A3 and specific for the fourth repetitive domain of human native Annexin A3, that is to say the capture and detection antibodies described above, are antibodies which exhibit a high affinity with an affinity constant of at least 10 ^-9 , preferably at least 10 ^-10 and which ^furthermore have a low dissociation constant of less than 2 10 ^-3 s ^-1 , more preferably lower at 5 10 ^-4 s ^-1 .

Preferably the antibodies are monoclonal antibodies and the preferred antibodies are the following antibodies: TGC42, TGC43 and TGC44 used as capture antibodies 13A12G4H2 and 1F10A6 used as detection antibodies. The preferred pair consisting of the TGC44 capture antibody and the detection antibody 13A12G4H2.

The invention also relates to an immunoassay kit for the in vitro diagnosis of prostate cancer in an urine sample to be analyzed comprising two antibodies, a capture antibody and a detection antibody, one of the two antibodies. being directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the other of the two antibody antibodies being directed against the fourth replicated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 2 and an explanatory note.

Preferably, the capture antibody is directed against the first repeated domain of native human Annexin A3 whose sequence is identified in SEQ ID NO: 1 and the detection antibody directed against the fourth repeated domain of Annexin A3. native human whose sequence is identified in SEQ ID NO: 2.

The capture and detection antibodies of the kit have the same characteristics as those described above for the process.

By antibody is meant a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody or a fragment of said antibodies, in particular Fab, Fab 'fragments, F (ab ') 2, ScFv, Fv, Fd. The requirement is that said antibodies must be specific for Annexin A3, that is to say that they do not cross-react with other annexins and that they are specific for the first repeated domain of Annexin A3 or specific to the fourth repeated domain of Annexin A3; the antibodies with the highest affinities and the weakest dissociation constants being the preferred antibodies.

The polyclonal antibodies can be obtained by immunizing an animal with the appropriate immunogen, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by chromatography. affinity on a column to which is attached an antigen specifically recognized by the antibodies.

The monoclonal antibodies can be obtained by the hybridoma technique whose general principle is recalled below.

Firstly, an animal, usually a mouse, is immunized with the appropriate immunogen whose B cells are then capable of producing antibodies against this antigen. These antibody-producing lymphocytes are then fused with "immortal" myeloma cells (murine in the example) to give rise to hybridomas. From the heterogeneous mixture of cells thus obtained, the cells capable of producing a particular antibody and of multiplying indefinitely are then selected. Each hybridoma is multiplied in the form of a clone, each leading to the production of a monoclonal antibody whose protein recognition properties can be tested, for example by ELISA, by immunoblot (Western blot) in one or two-dimensional, in immunofluorescence, or with a biosensor. The monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique described above.

The monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.

The capture antibody is preferably fixed, directly or indirectly, on a solid support, for example a cone, a well a microtitration plate etc ...

• les enzymes qui produisent un signal détectable par exemple par colorimétrie, fluorescence, luminescence, comme la peroxydase de raifort, la phosphatase alcaline, la β-galactosidase, la glucose-6-phosphate déshydrogénase,
• les chromophores comme les composés fluorescents, luminescents, colorants,
• les molécules fluorescentes telles que les Alexa ou les phycocyanines,
• les molécules radioactives comme le ³²P, le ³⁵S ou le ¹²⁵I.

The detection antibody is labeled with a marker reagent capable of directly or indirectly generating a detectable signal. A nonlimiting list of these marker reagents consists of:

• enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase,
• chromophores such as fluorescent compounds, luminescent compounds, dyes,
• fluorescent molecules such as Alexa or phycocyanines,
• radioactive molecules such as ³² P, ³⁵ S or ¹²⁵ I.

Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand. The ligand / anti-ligand couples are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin. In this case, it is the ligand that carries the binding partner. The anti-ligand can be detectable directly by the labeling reagents described in the preceding paragraph or be itself detectable by a ligand / anti-ligand.

These indirect detection systems can lead, under certain conditions, to amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference can be made to prior patent applications. FR2781802 or WO-A-95/08000 of the Applicant.

Depending on the type of labeling used, those skilled in the art will add reagents allowing the visualization of the marking.

The method of the invention is a "sandwich" type immunoassay carried out on a urine sample, in particular an "expressed" urine sample, that is to say a urine sample obtained after digital rectal examination.

The invention will be better understood with the aid of the following examples given for illustrative purposes, as well as with the aid of the appended figures.

FIGURES

• The figure 1 corresponds to the graphs relating to the ELISA Annexin A3 assay in ng / mL, in the urine expressed after the digital rectal examination, in 6 patients (Patients A to F), individually assayed with each of the sandwich ELISA assay formats whose name is indicated on the abscissa. The numerical values represented on this graph are presented in Table 4;
• the figure 2 represents the ELISA Annexin A3 assay during the analysis by successive filtrations (on a filter of 0.45 μm, then of 0.22 μm, then of 0.02 μm) of eight urine expressed after the digital rectal examination. The analysis was performed within 3-4 hours after sample collection. 5C5B10 assay panel: The samples and their fractions were assayed using the TGC44 / 5C5B10 ELISA. 13A12G4H2 assay panel: The samples and their fractions were assayed using the TGC44 / 13A12G4H2 ELISA. NF: unfiltered, control; FT: filtrate with the cut-off point of the filter used indicated. For each group of 8 fractions, the horizontal line represents the average of the 8 measurements observed. The doses of Annexin A3 are expressed as a% of the starting dose;
• the figure 3 represents the ELISA assay of Annexin A3 during the analysis by successive centrifugations (800 g, then 12,000 g, 150,000 g) of eight urine expressed after digital rectal examination. The analysis was performed within 3-4 hours after sample collection. 5C5B10 assay panel: The samples and their fractions were assayed using the TGC44 / 5C5B10 ELISA. 13A12G4H2 assay panel: The samples and their fractions were assayed using the TGC44 / 13A12G4H2 ELISA. NC: non-centrifuged, control; SN: supernatant with the indicated centrifugation rate. For each group of 8 fractions, the horizontal line represents the average of the 8 measurements observed. The doses of Annexin A3 are expressed as a% of the starting dose;
• the figure 4 represents the ELISA assay of Annexin A3 in the successive centrifugation assay (800 g , 12,000 g, 150,000 g) of ten other urine expressed after digital rectal examination. The analysis was performed within 3-4 hours after sample collection. 5C5B10 assay panel: The samples and their fractions were assayed using the TGC44 / 5C5B10 ELISA. 13A12G4H2 assay panel: The samples and their fractions were assayed using the TGC44 / 13A12G4H2 ELISA. % soluble = dose in ultracentrifugation supernatant / initial dose x 100; % exosome = (supernatant 12,000 g / initial dose x 100) -% soluble; % membrane = 100 - (supernatant 12,000 g / initial dose x 100).
• the figure 5 represents the reactivity of the 4 repeated D1 to D4 domains of Annexin A3, expressed in a recombinant form, with the 6 anti-Annexin A3 monoclonal antibodies indicated and analyzed by the Western blot technique. For the TGC42 and 1F10A6 gels, the first well corresponds to the molecular weight marker;
• the figure 6 is an alignment of the sequences of the different recombinant proteins expressing Annexin A3 domains and recapitulates the immunoreactivity of each of these recombinants with the monoclonal antibody TGC44, analyzed by the Western blot technique. The three points of suspension at the N-terminus or C-terminus of a sequence indicate that it continues in the construction, even though it is not shown entirely in the figure. The stop codon is represented by a star (*);
• the figure 7 illustrates the impact of Alanine mutations of the D4 domain on the recognition of ANXA3 by the 13A12G4H2 and 1F10A6 antibodies. The analysis was done by the ELISA technique. The capture antibody is TGC44. The 13A12G4H2, 1F10A6 antibodies tested were used in detection. The position of Alanine mutations on the protein sequence of ANXA3 is represented as abscissa. The ordinate is the "fold change signal" which corresponds to log ₂ (mutated protein signal / non-mutated protein signal). The 5C5B10 antibody that is not directed against the D4 domain is not impacted, it serves as a control. The arrows indicate the mutations for which there is a disturbance of the recognition signal for 13A12G4H2 and 1F10A6, which make it possible to define the residues involved in the binding of these antibodies.
• the figure 8 illustrates the absence of cross-reactivity of the ELISA assay formats described with 7 other proteins of the annexin family. The graph represents the ELISA signal obtained in Relative Fluorescence Values (RFV) on the VIDAS® apparatus for each of the test formats TGC44 / 5C5B10 and TGC44 / 13A12G4H2 and for each annexin indicated on the abscissa. By way of comparison, under the experimental conditions used, Annexin A3 made it possible to obtain a signal greater than 6000 RFV with each of the test formats;
• the figure 9 represents the sensorgrams obtained for each of the 6 monoclonal antibodies characterized using Biacore ™ T100. The graphs represent the resonance signal measured in "Resonance Units" (RU) as a function of time. Each curve in a graph represents all the measurements taken for a given Annexin A3 concentration. For each antibody 9 dilutions between 0 and 64 nM Annexin A3 were analyzed and are shown;
• the figure 10 groups together graphs relating to the ELISA Annexin A3 assay in urines expressed after digital rectal examination, in patients with biopsy-confirmed prostate cancer (cancer), and patients with prostate pathology but whose malignancy was excluded (not cancer). The group "not cancer" consists mainly of patients with benign prostatic hypertrophy. The graphs on the left of the page titled 5C5B10_SG represent the dose of Annexin A3 measured by the VIDAS TGC44 / 5C5B10 assay in ng / mL and normalized by the urinary density measured by the Combur ™ 10 strip according to the formula: normalized dose = dose VIDAS / (urinary density - 1). Similarly, the graphs on the right of the page labeled 13A12G4H2_SG represent the dose of Annexin A3 measured by the VIDAS TGC44 / 13A12G4H2 assay in ng / mL and normalized by urinary density. Two different cohorts of patients were studied: cohort # 1 has 127 patients and cohort # 2 has 94 patients. For each series of data, the series mean is represented as a horizontal line on the graphs. The probability associated with the unilateral Mann-Whitney test is also indicated on each graph: p-value < 0.05 = *, p-value <0.01 = **, ns = not significant.
• the figure 11 represents the effect of calcium ion and EDTA chelator on the doses measured with the 5C5B10 and 13A12G4H2 assays. Eleven urine collected after digital rectal examination were dosed directly (without treatment) or after addition of 5 or 25 mM CaCl ₂ or after addition of 5 or 25 mM EDTA. The y-axis represents the ratio of the doses with treatment (Ca ₂₊ or EDTA) / dose without treatment. The median of the series is represented as a horizontal line on the graphs. The probability associated with the Wilcoxon signed-rank test comparing the ratio to 1, this probability having been corrected for multiple tests according to Bonferroni, is also indicated on each series.

EXAMPLES Example 1: Detection of Annexin A3 by ELISA in Different Types of Samples Obtaining monoclonal antibodies

Immunization experiments were performed in female BALB / c (H-2 ^d ) mice six to eight weeks old at the time of the first immunization. Native human Annexin3 protein was purchased from Arodia Arotech Diagnostic (Cat No. 25592) and is purified from human neutrophils. This protein was mixed volume for volume with Freund's adjuvant (Sigma), prepared as a water-in-oil emulsion and which is known to have good immunogenicity. They received three successive doses of 10 μg of immunogen at zero, two and four weeks. All injections are performed subcutaneously. The first injection is made in admixture with the complete Freund's adjuvant, the next two are mixed with the incomplete Freund's adjuvant. Between D50 and D70 after the first injection, the humoral responses were restimulated with an intravenous injection of 100 μg of the native protein.

In order to follow the appearance of the antibodies, blood samples are regularly taken from the mice. The presence of anti-ANXA3 antibodies is tested using an ELISA. The protein of interest is used in capture (1 μg / well), after saturation antigen is reacted with different dilutions of test sera (incubation at 37 ° C for 1h). The specific antibodies present in the serum are revealed by a goat anti-mouse IgG AffiniPure conjugated to phosphatase alkaline (H + L, Jackson Immunoresearch, Cat No. 115-055-146), which binds to the desired antibodies (0.1 μg / well).

Three days after the last injection, one of the immunized mice was sacrificed; blood and spleen were removed. Splenocytes obtained from the spleen were cultured with Sp2 / 0-Ag14 myeloma cells to fuse and immortalize, according to the protocol described by (8, 9). After a 12-14 day incubation period, the resulting hybridoma supernatants were screened for the presence of anti-ANXA3 antibodies using the ELISA test described in the previous paragraph. The immunogen (native ANXA3), recombinant Annexin A3 produced in E. coli, and various human cells expressing ANXA3 were used successively to screen the hybridoma supernatants. Positive hybridoma colonies were subcloned twice according to the limiting dilution technique, well known to those skilled in the art.

The anti-annexin monoclonal antibodies A3, 5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6 were thus obtained.

Selection of anti-ANXA3 monoclonal antibodies for immunoassay assay of ANXA3

• Des cônes ont été sensibilisés avec un des anticorps de capture à tester, TGC42, TGC43, TGC44 ou 9C6B4 à une concentration de 10 µg /mL.
• Le contenu du deuxième puits de la cartouche HBs Ag Ultra a été remplacé par 300 µL d'anticorps de révélation à tester (5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6, TGC42, TGC43, TGC44), couplés à la biotine, dilués à 1µg/mL dans le tampon du deuxième puits du kit Vidas® HBs Ag Ultra (puits X1) contenant du sérum de chèvre et de l'azodure de sodium à 1g/L.

• La protéine ANXA3 native est testée diluée à 100, 25 et 3 ng/mL dans du tampon PBS. L'échantillon est déposé (150 µL) dans le premier puits (puits X0) de la cartouche HBs Ag Ultra.
• La réaction ELISA a été réalisée à l'aide de l'automate Vidas® et du protocole décrit pour le test HBs Ag Ultra.
• Les résultats ont été obtenus sous formes de valeurs brutes après soustraction du bruit de fond. Le signal est en RFV (relative fluorescente value).

The complementarity of the different anti-ANXA3 antibodies obtained as described above and the TGC42, TGC43 and TGC44 antibodies described in the patent application WO 2010/034825 was analyzed using as antigen the native ANXA3 (immunogenic) diluted in PBS buffer, performing in sandwich immunoassay. This type of assay can be performed in microplate, automated or manual, or by using automated immunoassays such as VIDAS (bioMérieux).
We used the reagents of the Vidas® HBs Ag Ultra Kit (bioMérieux, Cat No. 30315), as described in the corresponding leaflet (Cat. EN-2005/5 ) and amended as follows:

• Cones were sensitized with one of the capture antibodies to be tested, TGC42, TGC43, TGC44 or 9C6B4 at a concentration of 10 μg / mL.
• The content of the second well of the HBs Ag Ultra cartridge was replaced by 300 μL of test revealing antibody (5C5B10, 13A12G4H2, 9C6B4, 6D9D10, 1F10A6, TGC42, TGC43, TGC44), coupled to biotin, diluted to 1 μg / ml in the buffer of the second well of the Vidas® HBs Ag Ultra Kit (well X1) containing goat serum and sodium azodide at 1 g / L.

• The native ANXA3 protein is tested diluted at 100, 25 and 3 ng / mL in PBS buffer. The sample is deposited (150 μL) in the first well (well X0) of the HBs Ag Ultra cartridge.
• The ELISA reaction was carried out using the Vidas® automaton and the protocol described for the HBs Ag Ultra test.
• The results were obtained as raw values after subtraction of the background noise. The signal is in RFV (relative fluorescent value).

Table 1 below summarizes the results obtained (RFV signal), with the different combinations of antibodies used in capture or detection, on three dilutions of the purified native annexin A3 neutrophil (100, 25 and 3 ng / mL ). Table 1 Biotinylated revelation antibody Capture antibodies clone Dilution ANXA3 ng / ml TGC42 TGC43 TGC44 9C6B4 TGC42 0 - 22 11 17 3 - 44 20 78 25 - 238 89 500 100 - 859 297 1837 TGC43 0 106 - 39 63 3 111 - 36 82 25 144 - 56 257 100 232 - 86 914 TGC44 0 21 9 - 12 3 32 19 - 19 25 98 89 - 74 100 303 309 - 261 9C6B4 0 51 42 33 - 3 65 122 39 - 25 201 744 117 - 100 590 3525 357 - 5C5B10 0 20 12 93 9 3 5244 3669 5870 39 25 10480 9874 10429 249 100 11310 11095 11168 1139 13A12G4H2 0 26 21 18 19 3 2298 676 3733 23 25 9655 8468 10208 21 100 11380 10995 11332 21 6D9D10 0 12 23 16 9 3 11 23 14 9 25 12 23 18 19 100 18 41 43 27 1F10A6 0 45 9 14 14 3 159 207 194 17 25 4014 6728 5680 13 100 10489 10931 10688 15

• Si RFV à 3 ng/mL < 1000 alors « - »
• Si RFV à 3 ng/mL > 1000 alors « + »
• Si RFV à 25 ng/mL > 3000 alors « ++ »
• Si (RFV à 100 ng/mL > 9000) et (3000 < RFV à 25 ng/mL < 9000) alors « +++ »
• Si RFV à 100 ng/mL et 25 ng/mL > 9000 alors « ++++ »

Tableau 2 *mAb de détection biotinylé *mAb de capture TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 6D9D10 TGC42 - - - - ++++ ++++ +++ - TGC43 - - - + ++++ +++ +++ - TGC44 - - - - ++++ ++++ +++ - 9C6B4 + - - - + - - - mAb = anticorps monoclonal Table 2 below summarizes the results obtained described in Table 1, but with a different reading grid, to facilitate analysis and interpretation:

• If RFV at 3 ng / mL <1000 then "-"
• If RFV at 3 ng / mL> 1000 then "+"
• If RFV at 25 ng / mL> 3000 then "++"
• If (RFV at 100 ng / mL> 9000) and (3000 <RFV at 25 ng / mL <9000) then "+++"
• If RFV at 100 ng / mL and 25 ng / mL> 9000 then "++++"

Table 2 * biotinylated detection mAb * capture mAb TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 6D9D10 TGC42 - - - - ++++ ++++ +++ - TGC43 - - - + ++++ +++ +++ - TGC44 - - - - ++++ ++++ +++ - 9C6B4 + - - - + - - - mAb = monoclonal antibody

As can be seen from the table above, there are 9 combinations of complementary monoclonal antibodies that make it possible to perform a sandwich-type ELISA assay of ANXA3, with a very satisfactory signal dynamics ("+++" and "+" pairs). +++ "). Other combinations of complementary monoclonal antibodies are possible, namely 9C6B4 / TGC42, 9C6B4 / TGC43 and 9C6B4 / 5C5B10, but these solutions are not sufficiently robust from an analytical point of view and do not have sufficient analytical sensitivity to be used in the urine.

In capture, the monoclonal antibodies TGC42, TGC43 and TGC44 have equivalent performances with respect to Annexin 3.

In detection, the monoclonal antibodies 5C5B10 and 13A12G4H2 also have equivalent performances with respect to Annexin 3, whereas the monoclonal antibody 1F10A6 gives satisfactory but weaker signals than with the monoclonal antibodies 5C5B10 and 13A12G4H2.

Selection of anti-ANXA3 monoclonal antibodies according to their ability to detect prostatic ANXA3

The complementarity and capacity of anti-ANXA3 antibodies to detect prostatic ANXA3 was analyzed using the ELISPOT technique. This is a variant of the ELISA technique that directly detects secretions from cells in culture. WPE1-NB26 human cells (ATCC Cat No. CRL-2852), derived from a prostatic epithelial line RWPE-1 (Cat ATCC No. CRL-11609) were chosen as the expression model of human ANXA3 produced. by the prostate. These cells were transformed into cancer cells by treatment with MNU (N-methyl-N-nitrosourea) (10). They are cultured in K-SFM medium (Gibco) supplemented with 5 ng / ml of EGF (Epidermal Growth Factor) and 0.05 mg / ml of BPE (Bovin Pituitary Extract). Lines are incubated at 37 ° C with 5% CO ₂ .

The monoclonal capture antibodies (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) were adsorbed on 96-well MultiScreen ™ HTS plates (Millipore, Cat No. MSIP4510) at a concentration of 1 μg / well in sterile PBS. overnight at 4 ° C. The plates are then washed with PBS and saturated with culture medium containing 10% fetal calf serum (FCS). In parallel, the cells are counted, then diluted and distributed at 1000 cells per well. The plates are incubated for 20 hours at 37 ° C. and 5% CO ₂ , and then emptied. The remaining cells are then lysed by treatment with ice water for 10 minutes. The plates are then washed with PBS containing 0.05% Tween-20. The biotinylated revelation antibodies (TGC42, TGC43, TGC44, 5C5B10, 13A12G4H2, 9C6D9, 1F10A6) are added at 0.1 μg / well diluted in PBS-1% BSA-0.05% Tween-20 and incubated for 2 hours at room temperature. room. After several washes, the spots are revealed by the addition of alkaline extravidine-phophatase (Sigma, Cat No. E2636) for one hour at room temperature. then bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium substrate (BCIP / NBT, Biorad, Cat No. 170-6432).

The secretion of Annexin A3 by WPE1-NB26 cells is qualitatively measured by the observer. The number of spots observed is plotted on a scale of - to ++++ (Table 3). The best solution for detecting prostatic Annexin A3 is to match one of TGC42, TGC43 or TGC44 with an antibody selected from 13A12G4H2 and 1F10A6. The selected antibodies can be used indifferently in capture or detection, the only condition is that one antibody is associated with each group.

The results are summarized in Table 3 below. Table 3 * biotinylated detection mAb * capture mAab TGC42 TGC43 TGC44 9C6B4 5C5B10 13A12G4H2 1F10A6 TGC42 - - - + ++ ++++ ++++ TGC43 - - - + ++ ++++ ++++ TGC44 - - - + ++ ++++ ++++ 9C6B4 - - - - - - - 5C5B10 - - - + - - - 13A12G4H2 ++++ ++++ ++++ + - - - 1F10A6 +++ ++++ +++ + - - - * mAab = monoclonal antibody Surprisingly, the annexin A3 secreted by a prostatic cancerous line is less well detected when an antibody selected from TGC42, TGC43 and TGC44 is combined in capture and the 5C5B10 antibody in detection. Now, with VIDAS ^® immunoassay performed on annexin A3 purified neutrophils such a difference between 5C5B10 and 13A12G4H2 antibody was not observed, as shown in Table 2.

Detection of Annexin A3 in urine expressed by patients after digital rectal examination

Six urine specimens expressed after the digital rectal examination were obtained and treated according to the method described in the patent WO2007 / 141043 and by (11). The average duration of the prostate palpation was 10 seconds instead of the 20 seconds indicated in the request WO2007 / 141043 .

The ANXA3 contained in these different biological samples was quantified with each of the nine selected ELISA assays. The biotinylated detection antibodies were diluted to 0.5 μg / ml for the 5C5B10 and 13A12G4H2 monoclonals, to 1 μg / ml for the 1F10A6 clone. For each ELISA assay, a calibration curve was established by assaying a concentration range of the purified native Annexin A3 protein (Arodia). The calibration curve was plotted by plotting on the abscissa the logarithm in the base ten of the concentration and on the ordinate the signal measured by Vidas ^® in RFV. The concentration of ANXA3 present in the biological sample was calculated by interpolating the concentration corresponding to the RFV signal read by the Vidas®, using mathematical nonlinear regression models such as a third-order polynomial or a 4- model. PL, well known to those skilled in the art. The results are shown in Table 4 below. Table 4 Annexin A3 doses in ng / mL according to ELISA assay format (Capture Antibody - Detection Antibody) P * TGC42-5C5B10 TGC43-5C5B10 TGC44-5C5B10 TGC42-13A12G4H2 TGC43-13A12G4H 2 TGC44-13A12G4H 2 TGC42-1F10A6 TGC43-1F10A6 TGC44-1F10A6 AT 90 99 123 0.1 0.1 0.1 0.1 0.1 0.1 B 5 2 5 9 11 13 13 14 14 VS 2 1 2 6 8 8 9 9 10 D 11 3 13 78 80 81 77 86 97 E 22 7 28 110 144 154 120 136 149 F 1100 810 2100 0.0 0.0 0.0 0.0 0.0 0.0 P * = patient

The results of the Vidas® ELISA assays for ANXA3 in all six patients are summarized in Table 4 and shown in the table below. Figure 1 . These results are extremely surprising and highlight a complexity of ANXA3 urinary hitherto unsuspected. Indeed, for a given sample, the 9 ELISA test formats do not always make the same dose. This difference is bound to the detection antibody: for a given sample and a given detection antibody, the rendered doses are very similar, or even identical regardless of the capture antibody (TGC42, TGC43 or TGC44). The correlation between the doses obtained by the formats using TGC42 and those obtained by the formats using TGC43 is 0.956 (Spearman r, p-value <0.0001). The correlation between the doses measured by the formats using TGC42 and that obtained by the formats using TGC44 is 0.994 (Spearman r, p-value <0.0001). The 3 capture antibodies behave very similarly. They are not the cause of the observed differences.

Annexin A3 concentrations calculated with ELISA using 13A12G4H2 or 1F10A6 antibodies were also correlated (Spearman's r = 0.999, p-value <0.0001). The slope of the line calculated by placing on the abscissa the doses obtained by the ELISA using 13A12G4H2 and on the ordinate those obtained by the ELISA using 1F10A6 is 1: these two antibodies make identical doses. Conversely, there is no correlation between the doses calculated with the 5C5B10 antibody used in detection and those calculated with 13A12G4H2.

Based on this analysis performed on expressed urine, the 9 ELISA assays can be divided into 2 groups. Group 1 corresponds to the combinations of capture antibodies TGC42, TGC43 or TGC44 with clones 13A12G4H2 or 1F10A6 used in detection. Group 2 corresponds to the combinations of capture antibodies TGC42, TGC43 or TGC44 with the 5C5B10 monoclonal used in detection. Since each of these antibodies is specific for ANXA3, both ELISA groups measure different biological information. The ELISPOT assay presented above suggests that Group 1 ELISA using 13A12G4H2 or 1F10A6 clones are more suitable for detecting prostatic ANXA3.

Example 2 Characterization of Urinary Annexin A3

Eight urine samples expressed after digital rectal examination were obtained according to the method described in Example 1, transported from the hospital to the laboratory and subjected to the analyzes described in the 4 hours following the collection. The TGC44 / 13A12G4H2 assay (called only 13A12G4H2 thereafter) was chosen as the assay Prototype Group 1 defined in Example 1. Similarly, TGC44 / 5C5B10 assay (called only 5C5B10 thereafter) was chosen as the prototype assay representing the group 2.

Analysis of urine expressed by successive filtrations

• Une première filtration sur une membrane avec des pores de 0,45 µm de diamètre (Millex), le filtrat est ensuite récupéré et un aliquot mis de côté pour dosages : FT 0,45 µm.
• Le filtrat FT 0,45 µm est filtré sur une membrane de 0,22 µm (Millex) et un aliquot mis de côté pour dosages : FT 0,22 µm.
• Le filtrat FT 0,22 µm est filtré de nouveau sur une membrane de 0,02 µm (Millipore) cette fois, dont la taille des pores retient les exosomes urinaires, c'est le FT 0,02 µm.

The freshly harvested urine has undergone successive filtrations:

• A first filtration on a membrane with pores of 0.45 μm in diameter (Millex), the filtrate is then recovered and an aliquot set aside for assays: FT 0.45 microns.
• The 0.45 μm FT filtrate is filtered on a membrane of 0.22 μm (Millex) and an aliquot set aside for assays: FT 0.22 μm.
• The 0.22 μm FT filtrate is filtered again on a membrane of 0.02 μm (Millipore) this time, the pore size of which retains the urinary exosomes, the FT 0.02 μm.

Each of these fractions is then assayed with the 2 prototype ELISA test formats on Vidas® PLC. The doses of Annexin A3 measured in each of the fractions are expressed as a percentage of the initial dose of urinary Annexin A3 before the first filtration (= 100%). The results are represented in the Figure 2 . The decrease in the dose between two successive filtrates indicates that biological particles containing ANXA3 were retained on the filtration membrane. Thus, according to principles well known to those skilled in the art, filtration on 0.45 microns retains cells and cellular debris (12, 13), a 0.22 micron filtration retains certain organelles and subcellular fractions and a filtration on 0.02 μm retains the exosomes (14-16). This decrease is well observed for all samples, with the two assay methods used (5C5B10 and 13A12G4H2). In the vast majority of urines expressed (6/8), approximately 20 to 40% of ANXA3 is associated with cellular debris or other subcellular fractions. The remaining 60 to 80% are removed by filtration over 0.02 μm, and are therefore associated with exosomes. Of the 8 tested urine specimens tested, soluble ANXA3 (which passes through a 0.02 μm filter) was found only in a single sample with the 5C5B10 assay and in two samples with the 13A12G4H2 assay. .

Analysis of urine expressed by successive centrifugations

• Les urines sont centrifugées une première fois à 800 g pendant 5 min, le surnageant est récupéré et un aliquot mis de côté pour dosages : SN 800.
• Le surnageant urinaire issu de la centrifugation à 800 g est centrifugé à 12 000 g pendant 7 min, le surnageant est récupéré et un aliquot mis de côté pour dosages : SN 12 000.
• Le surnageant urinaire obtenu après centrifugation à 12 000 g est ensuite ultracentrifugé à 150 000 g sur la nuit à 4°C afin de précipiter les exosomes urinaires dans le culot. Le surnageant récolté est le SN ultra.

The same freshly collected urine was also fractionated by successive centrifugations:

• The urine is centrifuged a first time at 800 g for 5 min, the supernatant is recovered and an aliquot set aside for assays: SN 800.
• The urine supernatant resulting from the centrifugation at 800 g is centrifuged at 12,000 g for 7 min, the supernatant is recovered and an aliquot set aside for assays: SN 12,000.
• The urine supernatant obtained after centrifugation at 12,000 g is then ultracentrifuged at 150,000 g overnight at 4 ° C to precipitate urinary exosomes in the pellet. The harvested supernatant is the ultra SN.

Each of these fractions is then assayed with the 2 prototype ELISA test formats on Vidas® PLC. The doses of Annexin A3 measured in each of the fractions are expressed as a percentage of the initial dose of urinary Annexin A3 prior to any centrifugation (NC = 100%). The results are represented in the Figure 3 . Decreasing the dose between two successive supernatants indicates that ANXA3-containing biological particles were separated in the pellet during centrifugation. Thus, according to principles well known to those skilled in the art, 800 g centrifugation culminates most human cells, a centrifugation at 12 000 g culminates residual cell debris and some organelles and ultracentrifugation on the night culminates all particles subcellular, including exosome-type vesicles (17, 18). Any protein that remains in the supernatant after ultracentrifugation is considered soluble (19). This decrease is well observed for all samples, with the two assay methods used (5C5B10 and 13A12G4H2). In the vast majority of urines expressed (6/8), approximately 20 to 25% of ANXA3 is associated with cellular debris or other subcellular fractions. The remaining 75 to 80% are precipitated only by ultracentrifugation at night, and are therefore associated with exosome-like particles. Of the 8 urine specimens tested, soluble ANXA3 (which remains in the supernatant after overnight ultracentrifugation) was found only in the two samples already identified as containing soluble ANXA3 with the analysis described in paragraph 1, with the two assay techniques 5C5B10 and 13A12G4H2.

A new series of 10 freshly harvested urine was analyzed by slightly modifying the centrifugation conditions. Centrifugation at 800 g was 10 min. and that at 12,000 g of 30 min. The results of this second experiment of successive centrifugations are presented in Figure 4 and confirm the observations made during the first experiment ( Figure 3 ). In addition, they show that the 13A12G4H2 assay recognizes more frequently than the 5C5B10 ANXA3 assay associated with exosomes.

Overall, the filtration and centrifugation fractionation results are extremely consistent and indicate that the majority of the ANXA3 to be assayed is associated with exosomes, but not only. We have shown that ANXA3 is also associated with larger particles such as cell debris, and that it can also be in soluble form. The sample preparation for a Western blot analysis makes it possible to solubilize and denature the proteins and thus to reduce this complexity, which can partly explain the difficulties encountered so far in finding an ELISA assay method that can be used in the biological fluids and in particular urine. Of course, the method of treatment of the expressed urine collected, the storage conditions (temperature, buffer) are all factors that can vary the distribution of ANXA3 in the various urinary fractions where it may be present.

Example 3 Determination of Epitopes Recognized by Anti-ANXA3 Monoclonal Antibodies Expression of the 4 annexin repeat domains of Annexin A3 in recombinant form and determination of the repeated domains recognized by the monoclonal antibodies.

D2 99-159 92-160

D3 183-243 171-245

D4 258-318 252-323

^a Réel : Etendue du domaine répété, du premier au dernier acide aminé, selon la base de données UniProtKB (http://www.uniprot.org).
^b Exprimé : Etendue de la construction contenant le domaine répété, numérotation des acides aminés selon UniProtKB. Les constructions contiennent quelques acides aminés supplémentaires aux extrémités N et C terminales du domaine, afin de ne pas interrompre les hélices alpha et leur permettre de se former. Like all the members of the Annexin family, Annexin A3 contains in its protein sequence domains called annexin repeat. These repeated domains are four in number and characterize the family. To determine the repeated domain recognized by each of the monoclonal antibodies, these domains were expressed in a recombinant form. A sequence of 8 histidines was added to the N-terminal part of each domain to allow purification by metal-chelate affinity chromatography. Table 5 summarizes the protein sequences of the recombinant constructs making it possible to express each of the repeated domains in isolation. Table 5 Field Real ^a Expressed ^b Protein sequence D1 27-87 19-89

D2 99-159 92-160

D3 183-243 171-245

D4 258-318 252-323

^a Real: Extent of repeated domain, from first to last amino acid, according to the UniProtKB database ( http://www.uniprot.org ).
^b Expressed: Extent of the construction containing the repeated domain, numbering of amino acids according to UniProtKB. The constructs contain some extra amino acids at the N- and C-termini of the domain, so as not to interrupt the alpha helices and allow them to form.

The nucleic acid sequences corresponding to the protein sequences of the D1, D2, D3 and D4 domains were obtained by chemical synthesis by Geneart. These nucleic sequences have been optimized to promote the expression of proteins in Escherichia coli. The DNA fragments were cloned between the Nco I and Xba I sites of the prokaryotic expression vector pMRCH79 (derived from pMR78, bioMérieux). The plasmids thus obtained were transformed into BL21 (DE3) bacteria (Stratagene). The cultures to produce the different domains are carried out at 37 ° C. with stirring in 2-YT medium (Invitrogen). Induction is performed with 0.5 mM IPTG (isopropyl beta-1-thiogalactosidase). The bacterial pellets are directly taken up in the sample buffer of the NuPAGE Novex gels (Invitrogen) following the procedure provided with the gels under reduced conditions. Protein separation is performed in NuPAGE Novex Bis-Tris 4-12% Gel. Western blot analysis of the reactivity of the monoclonal antibodies for the different domains of annexin A3 is produced using a chemiluminescent substrate, according to the process described in the patent application WO 2009/019365 for example, well known to those skilled in the art. The antibodies to be tested were used at a dilution of 10 μg / ml. The exposure time was 100 seconds unless otherwise indicated.

Each anti-ANXA3 antibody was tested with recombinants expressing the 4 repeated ANXA3 domains; the results of this Western blot analysis are presented in the Figure 5 . The monoclonal antibodies TGC42, TGC43, TGC44 and 5C5B10 are specific for the D1 domain. Monoclonal antibodies 13A12G4H2 and 1F10A6 are directed against the D4 domain. As for the 9C6D4 and 6D9D10 antibodies, they do not recognize any of the 4 repeat domains, which most likely indicates that their epitopes are located outside the repeated domains of ANXA3.

Fine analysis of epitopes recognized by anti-ANXA3 antibodies

The determination of epitopes has been made with the Spotscan technique according to Frank and Döring (20), which is described in detail in the patent application. WO 2009/019365 . For this purpose, the entire protein sequence of Annexin A3 was reproduced on a nitrocellulose membrane in the form of peptides of 12 overlapping amino acids, shifted by 2 amino acids. Then in a second synthesis, the ANXA3 sequence was reproduced as overlapping amino acid peptides, staggered with an amino acid. The immunoreactivity of these overlapping peptide membranes was tested with anti-ANXA3 antibodies.

Thus, it has been possible to more finely delineate anti-ANXA3 monoclonal antibody epitopes among the 8 studied. The epitopes deduced from the comparison of the sequences of the recognized overlapping peptides are summarized in Table 6. The minimal epitope is the minimum sequence necessary to have a recognition of the antibody, with a more or less intense signal. The optimal epitope is the ideal sequence for the best possible recognition of the antibody, including or identical to the minimal epitope. Our results confirm that TGC42 and TGC43 are well directed against a single epitope, the one that is originally described in the application WO 2010/034825 . Surprisingly, the 5C5B10 antibody defines a new epitope that was not described in the prior art. The antibodies 6D9D10 and 9C6D4 are specific for the N-terminus of the protein, as the monoclonal TGC7 of the application WO 2007/141043 . The monoclonal antibodies TGC44, 13A12G4H2 and 1F10A6 show no Spotscan reactivity, even on a membrane carrying peptides 20 amino acids long. They probably have conformational or at least semi-conformational epitopes whose structures are not sufficiently well reproduced by synthetic peptides. Table 6 Antibody Field Optimal epitope ^has Minimal epitope ^b TGC42 D1 SNAQRQLIVKEYQAAYG (49-65) (SEQ ID: 10) LIVKEYQAAYG (55-65) (SEQ ID: 11) TGC43 D1 IVKEYQAAYGKE (56-67) (SEQ ID: 12) KEYQAAYG (58-65) (SEQ ID: 13) 5C5B10 D1 DLSGHFEHL (75-83) (SEQ ID: 14) LSGHFEH (76-82) (SEQ ID: 15) 6D9D10 N-term ASIWVGHRGTVRDYPDFSPS (2-21) (SEQ ID: 16) SIWVGHRGTVRDYPDFSP (3-20) (SEQ ID: 17) 9C6D4 N-term YPDF (15-18) (SEQ ID: 18) YPDF (15-18) SEQ ID: 18) ^a Optimal epitope: Ideal sequence allowing the best possible recognition of the antibody (including or identical to the minimal epitope).
^b Minimum epitope: Minimum sequence necessary to have recognition of the antibody (more or less intense signal).

Accurate localization of the TGC44 antibody epitope by the Novatope technique

The Novatope system (Merck, Cat No. 69279) is a technology for the analysis of a protein for the purpose of selecting domains containing epitopes. The method is based on the creation of a library of bacterial clones, each expressing a fragment of the protein, cut randomly. These clones are analyzed by immunorevealing with the antibody that is to be characterized. DNA sequencing of the positive clones makes it possible to deduce the protein sequence from a fragment containing the epitope. The technique was applied following the procedure provided with the kit.

Thus, two clones reacting with the TGC44 antibody could be isolated and sequenced. Clone 2J7 expresses the sequence KEYQAAYGKELKDDLKGDLSGHFEHLMVALVTPPAVFD (SEQ ID: 19) which corresponds to residues 58-95 of ANXA3. Clone 2Z13 expresses the sequence QKAIRGIGTDEKMLISILTERSNAQRQLIVKEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID: 20) which corresponds to residues 28-83 of ANXA3. The common part between the sequences of these two clones are the residues 58-83 of Annexin A3, that is the sequence KEYQAAYGKELKDDLKGDLSGHFEHL (SEQ ID: 21). In addition, the TGC44 and 5C5B10 antibodies being complementary (see Example 1), their two epitopes can not be overlapping, one can thus remove the amino acids corresponding to the epitope of the antibody 5C5B10, or DLSGHFEHL (75-83) ( SEQ ID: 14). Thus, the epitope of the TGC44 antibody is included in the sequence KEYQAAYGKELKDDLKG (58-74) (SEQ ID: 22). This is the same region as that recognized by TGC42 and TGC43. On the other hand, the fact that the TGC44 antibody does not exhibit Spotscan reactivity indicates a conformational constraint for the recognition. TGC44 is able to bind to its epitope only when the sequence KEYQAAYGKELKDDLKG (58-74) (SEQ ID: 22) is fused on the N-terminal side to a long sequence of at least 30 residues. Thus, the recombinant repetitive domain D1, whose sequence is identified in SEQ ID NO: 6, clones 2J7 and 2Z13 fused to a carrier protein or the recombinant fragment vANA-7 described requires WO 2010/034825 are all recognized by clone TGC44. In contrast, the recombinant fragment vANA-3, which lacks the first 34 N-terminal amino acids, is not recognized ( Figure 6 )

Accurate localization of the 13A12G4H2 and 1F10A6 antibody epitope

The experience presented in Figure 5 shows that the epitopes of the 13A12G4H2 and 1F10A6 antibodies are contained in the D4 domain of annexin A3. Twelve recombinant proteins were constructed by site-directed mutagenesis to enhance the "mapping" of antibodies against the D4 domain. This is the complete sequence of mature native ANXA3 (aa 2-323), fused on the N-terminal side to a histidine tag (unmutated sequence). PCR mutagenesis was used to introduce Alanine mutations at positions 253, 257, 260, 263, 265, 268, 270, 274, 306, 311 and 317 of the protein sequence (GeneArt Mutagenesis Service, Invitrogen). This technique known as Alanine scanning allows to evaluate one by one the importance of the contribution of each mutated amino acid residue of Annexin A3 to the binding of antibodies 13A12G4H2 and 1F10A6. All these DNA fragments were cloned into the vector pMRCH79 derived from the vector pMR78, then transformed into BL21 bacteria (DE3). The production and purification of the proteins were carried out according to the techniques well known to those skilled in the art and which were mentioned at the beginning of Example 3.

These 12 proteins (11 mutants and 1 non-mutated control) were then used to evaluate the binding capacity of the 13A12G4H2 and 1F10A6 antibodies in a sandwich ELISA, using the TGC44 antibody in capture. The 5C5B10 detection antibody whose epitope is in the D1 domain and therefore should not be affected by the D4 domain mutations is used as a control. The results are presented in the Figure 7 . The graph represents the "fold change signal" (on the ordinate) of each mutation (on the abscissa). The "fold change signal" corresponds to log ₂ (mutated protein signal / non-mutated protein signal). The higher the absolute value of the fold change signal, the more the mutations for which this variation is observed affect the binding of the antibody. Thus, for 5C5B10, the fold change signal is always around 0, indicating that none of the mutations tested disturb the binding of this monoclonal. On the other hand, for 13A12G4H2 and 1F10A6, it was possible to identify amino acids whose mutation prevents or disrupts the fixation very significantly. These are positions 260, 263, 265 and 306. The mutation of position 270 has a measurable but less significant impact than for the previous positions. This shows that the amino acids 260, 263, 265 and 306 of ANXA3 belong to the epitope recognized by the monoclonal antibodies 13A12G4H2 and 1F10A6, the two most important residues in the antigen-antibody interaction being Lysine (K). position 263 and Aspartic Acid (D) at position 306.

Example 4 Analytical Specificity of Anti-ANXA3 ELISA Assay Formats

Annexins are a family of proteins that share function and sequence homologies. A BLAST query performed on the UniProtKB database, reduced to sequences of human origin, has made it possible to identify the proteins having the greatest sequence homology with Annexin A3. These are, in descending order of homology, annexin A4, annexin A11, annexin A6 and annexin A5.

Therefore, it was important to demonstrate the specificity of ELISA assays for Annexin A3 and the absence of cross-reactivity with other family members. Since the 3 antibodies TGC42, TGC43 and TGC44 are directed against the same epitope, the TGC44 / 13A12G4H2 assay (hereinafter called only 13A12G4H2) was chosen as the prototype group 1 assay defined in Example 1. In the same way, the TGC44 / 5C5B10 assay (hereinafter only referred to as 5C5B10) was chosen as the prototype assay representing group 2. The lack of cross-reactivity was tested using commercially available antigens obtained from Abnova: annexin A1 (Cat No. H00000301-P01), Annexin A2 (Cat No. H00000302-P01), Annexin A4 (Cat No. H00000307-P01), Annexin A5 (Cat No. H00000308-P01), Annexin A6 (Cat No. H00000309- P01) and annexin A11 (Cat No. H00000311-P01). Annexin A13 was expressed in recombinant form in the laboratory by cloning into the pMRCH79 expression vector; fused to a histidine tag, and then purified by metal-chelate affinity chromatography. The proteins were diluted in the VIDAS well X1 buffer at a concentration of 12.5 μg / mL for TGC44 / 5C5B10 ELISA and at a concentration of 16 μg / mL for TGC44 / 13A12G4H2 ELISA. The results are presented in the Figure 8 . Both TGC44 / 13A12G4H2 and TGC44 / 5C5B10 ELISA formats are both highly specific for Annexin A3 and show no cross-reactivity with the other annexins tested.

Example 5 Determination of the Affinity of Anti-Annexin A3 Antibodies

Surface plasmon resonance technology allows real-time visualization of interactions between various unlabeled biomolecules. One of the reagents is specifically attached to a sensor chip while the other species involved in the interaction is in a continuous stream of buffer. Surface plasmon resonance measurements have been performed using a Biacore T100. The reagents including the CM5 sensor chip, anti-mouse IgG, specific for the Fc fragment, obtained in the rabbit (RAM Fc), and the coupling kit via the amines for the immobilization of the antibodies were all obtained from GE -Healthcare Bioscience AB.

In order to study the kinetic binding characteristics of the anti-Annexin A3 antibodies, the latter were immobilized by capture on the sensor chip, on which the RAM Fc antibody had been previously covalently coupled. The binding experiments were performed in buffer, HEPES, at 25 ° C, with a flow rate of 30 μl / min. The modification of the resonance signal in RU (Resonance Unit) makes it possible to follow in real time the fixation and the dissociation of the biomolecules on the surface of the biosensor. Initially, the monoclonal antibody to be studied was injected into channel 2 to obtain a signal of approximately 250 RU. Subsequently, Annexin A3 (Arodia) was injected into channels 1 and 2. The association and dissociation times were respectively 5 and 15 minutes. After measuring the resonance responses, the surface of the biosensor was regenerated by washing with 50 mM HCl at 10 μL / min for 120 seconds. The same measurement method was repeated for each dilution of the Annexin A3 protein; in total 9 different protein dilutions between 0 and 64 nM were analyzed. The resulting sensorgrams were plotted and analyzed with the dedicated Biacore ™ T100 software, according to the 1: 1 interaction model. The kinetic constants of association (Kon) and dissociation (Koff) were measured using the antibodies at a concentration of 3 μg / mL, except for 13A12G4H2 and 1F10A6 which were used at 0.75 μg / mL in order to limit the impact of background noise. The affinity, represented by the dissociation constant (Kd), was calculated (Kd = Koff / Kon).

For each anti-Annexin A3 antibody, the graphs representing the resonance signal as a function of time are presented in FIG. Figure 9 . The measured values of the association and dissociation constants, as well as the calculated value of the affinity constants are shown in Table 7.

The set of anti-ANXA3 antibodies studied has high affinities, with Kd values ranging from 10 ^-9 to 5 x 10 ^-10 M. The antibodies can be classified into two distinct groups. function of their Kd. The first group corresponds to antibodies 13A12G4H2, TGC42 and TGC44 whose Kd is greater than 10 ^-10 M, it is the group of very high affinity antibodies. The second group corresponds to the antibodies 5C5B10, 1F10A6 and TGC43 whose Kd is between 10 ^-9 and 3.5 x 10 ^-9 M, it is the group of high affinity antibodies.

The three antibodies used in capture, TGC42, TGC43 and TGC44, have equivalent kinetic association constants, and therefore equivalent capture capacities. The 13A12G4H2 antibody also has a comparable association kinetic constant, thus confirming its capture capacity shown in Example 1. The kinetic constant of the 5C5B10 antibody is approximately 1 log lower than that of TGC44. , which illustrates its lower catch capacity.

With regard to the kinetic dissociation constants, it is also possible to divide the anti-ANXA3 antibodies into 2 groups. The first group contains TGC44, TGC42, 13A12G4H2 and 5C5B10; it is characterized by a very low dissociation kinetic constant, between 9 x 10 ^-5 and 3.5 x 10 ^-4 s ^-1 . Once antigen-antibody binding has occurred, Annexin A3 is retained by the antibodies in this group and does not dissociate. The second group contains TGC43 and 1F10A6; it is characterized by a higher dissociation kinetic constant, of the order of 10 ^-3 s ^-1 . Antibodies in this group, even if they succeed in attaching Annexin A3, dissociate much more rapidly. Thus, although having comparable kinetic association constants, TGC42 and TGC44 are better capture antibodies to be used for the development of an ELISA assay than TGC43. Table 7 Antibody fixed on the biosensor Kon (M ^-1 .s ^-1 ) Koff (s ^-1 ) Kd (M) 13A12G4H2 5,3E + 05 2.7E-04 5,0E-10 TGC42 7,3E + 05 1.0E-04 1,8E-10 TGC44 9,5E + 05 9,7E-05 1.0E-10 5C5B10 9,8E + 04 3.4E-04 3.4E-09 1F10A6 3,8E + 05 1,1E-03 2,9E-09 TGC43 8,4E + 05 1,1E-03 1.3E-09

Example 6 Use of Prototype ELISA Assays to Differentiate Cancer and Not Cancer in Patient Cohorts

In order to analyze the ability of Annexin A3 to distinguish patients with prostate cancer from those who do not, the ELISA assays described in Example 1 were used to assay the amount of Annexin A3. present in the urine post-rectal examination of these patients.

The patients included in the "Cancer" group all have proven prostate cancer, whose diagnosis was confirmed by histological analysis on a biopsy. For patients included in the "No cancer" or control group, prostate cancer was excluded by biopsy as well; the vast majority are patients with benign prostatic hyperplasia. The collection and treatment of post-digital rectal examination urine samples were carried out according to the method described in Example 1. Two cohorts of patients were analyzed: Cohort # 1 comprises 127 patients, 72 in the "Cancer" group and 55 in the "Non cancer" group. Cohort # 2 includes 94 patients, 43 in the "Cancer" group and 42 in the "Non cancer" group. For both cohorts, these are patients with serum PSA between 2.5 and 10 ng / mL. This is the gray area of PSA, in which the clinical performance of the marker is the worst.

The first ELISA assays carried out on the urine expressed after the digital rectal examination, described in Example 1, made it possible to highlight a biological complexity that was previously unsuspected. We have succeeded in defining two groups of ELISA assays that measure different biological information (partially redundant or not). It is therefore essential to determine which group of ELISA assays gives access to the most relevant biological information, ie it makes it possible to better discriminate between "Cancers" and "No cancers" in a given sample. The TGC44 / 13A12G4H2 assay (hereinafter called 13A12G4H2 only) was chosen as the prototype group 1 assay defined in Example 1. Similarly, the TGC44 / 5C5B10 assay (referred to only as 5C5B10 thereafter) was chosen as a prototype assay representing group 2. The Figure 10 represents the values obtained for each cohort # 1 and # 2 with both formats Annexin A3 ELISA assay used. The dose of Annexin A3 was normalized to the urinary density value measured by the Combur 10 strip (Roche Cat No. 04510062171), according to the standardized dose formula = VIDAS / dose (urinary density-1) (21). ).

In the first cohort of patients (cohort # 1) the two assay formats make it possible to discriminate cancer patients from controls. In fact, the standardized doses of Annexin A3 are significantly lower in the "Cancer" group than in the "Non cancer" group, regardless of the dosage format used (unilateral Mann-Whitney p-value is 0.003 for the TGC44 / 5C5B10 assay and 0.02 for the TGC44 / 13A12G4H2 assay). On the other hand, in the second cohort (cohort # 2), it is only the TGC44 / 13A12G4H2 assay format that makes it possible to discriminate patients with cancer from controls. As for cohort # 1 and in agreement with the Western blot study by Schostack et al. (21), standardized Annexin A3 doses were significantly lower in the "Cancer" group than in the "Non cancer" group using the prototype TGC44 / 13A12G4H2 ELISA (one-sided Mann-Whitney p-value was 0.01) . On the other hand, the prototype TGC44 / 5C5B10 is taken by surprise on this second cohort and does not make it possible to discriminate the patients having a cancer. The prototype TGC44 / 13A12G4H2 and, more generally, the Group 1 ELISA formats thus seem to be superior in discriminating prostate cancers. This conclusion is consistent with the ELISPOT assay described in Example 1 which suggests that Group 1 ELISA is more suitable for detecting prostatic ANXA3.

Example 7 Effect of Calcium Ion and EDTA on Assays 5C5B10 and 13A12G4H2

Eleven urine collected after the digital rectal examination were dosed directly (without treatment) or after addition of 5 or 25 mM CaCl ₂ or after addition of 5 or 25 mM EDTA. The results are presented in Figure 11 . The y-axis represents the ratio of the doses with treatment (Ca ₂₊ or EDTA) / dose without treatment, for the 5C5B10 assay and the 13A12G4H2 assay. The addition of the calcium ion in the urine reduces the doses measured by the 13A12G4H2 assay and this in a progressive manner, plus the added calcium concentration is increased, the lower the measured dose. The 5C5B10 assay is much less affected by the presence of calcium in the urine, although at high calcium concentration a slight effect is observed. As for the EDTA treatment of the urine, it has no effect on the 13A12G4H2 assay but for the 5C5B10 assay, it causes a very slight increase in the measured doses. The introduction of EDTA or a similar chelating agent into the urine before dosing, via the buffers or in solid form, formulated or not, thus makes it possible to improve the assays, in particular the 13A12G4H2 assay by making it possible to reduce the effect of calcium ion on doses of ANXA3.

Bibliographical references

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2. 2. Barton, GJ, Newman, RH, Freemont, PS, and Crumpton, MJ, Amino acid sequence analysis of the annexin super gene family of proteins, Eur J Biochem, 198, 749 (1991). ).
3. 3. Cabec, V., Russo-Marie, F., and Maridonneau-Parini, I. Differential expression of two forms of annexin 3 in human neutrophils and monocytes and their differentiation, Biochem Biophys Res Commun, 189, 1471 (1992). ).
4. 4. Cabec, V., and Maridonneau-Parini, I., Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translocates to the plasma membrane in activated cells, Biochem J, 303 (Pt 2), 481 (1994). ).
5. 5. Bianchi, C., Bombelli, S., Raimondo, F., Torsello, B., Angeloni, V., Ferrero, S., Di Stefano, V., Chinello, C., Cifola, I., Invernizzi, L. , Brambilla, P., Magni, F., Pitto, M., Zanetti, G., Mocarelli, P., and Perego, RA, Primary cell cultures from human renal cortex and renal-cell carcinoma isoforms of Annexin A3, Am J Pathol, 176, 1660 .
6. 6. Kollermann, J., Schlomm, T., Bang, H., Schwall, GP, von Eichel-Streiber, C., Simon, R., Schostak, M., Huland, H., Berg, W., Sauter, G. Klocker, H., and Schrattenholz, A., Expression and prognostic relevance of annexin A3 in prostate cancer, Eur Urol, 54, 1314 (2008). ).
7. 7. Wozny, W., Schroer, K., Schwall, GP, Poznanovic, S., Stegmann, W., Dietz, K., Rogatsch, H., Schaefer, G., Huebl, H., Klocker, H., Schrattenholz , A., and Cahill, MA, Differential radioactive quantification of protein abundance ratios between benign and malignant prostate tissues: cancer association of annexin A3, Proteomics, 7, 313 (2007) ).
8. 8. Kohler, G., and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256, 495 (1975). ).
9. 9. Kohler, G., Howe, SC, and Milstein, C., Fusion between immunoglobulin-secreting and nonsecreting myeloma cells, Eur J Immunol, 6, 292 (1976). ).
10. 10. Webber, MM, Quader, ST, Kleinman, HK, Bello-DeOcampo, D., Storto, PD, Bice, G., DeMendonca-Calaca, W., and Williams, DE, Human cell lines as in vitro / in vivo model for prostate carcinogenesis and progression, Prostate, 47, 1 (2001) ).
11. 11. Rehman, I., Azzouzi, AR, Catto, JW, Allen, S., Cross, SS, Feeley, K., Meuth, M., and Hamdy, FC, Proteomic analysis of voided urine after prostatic massage from patients with prostate cancer : a pilot study, Urology, 64, 1238 (2004) ).
12. 12. Clark, HF, Geldreich, EE, Jeter, HL, and Kabler, PW, The membrane filter in sanitary bacteriology, Public Health Rep, 66, 951 (1951 ).
13. 13. Kabler, PW, and Clark, HF, The Use of Differential Media with the Membrane Filter, Am J Health Public Health Nations, 42, 390 (1952 ).
14. 14. Cheruvanky, A., Zhou, H., Pisitkun, T., Kopp, JB, Knepper, MA, Yuen, PS, and Star, RA, Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator, J Physiol Renal Physiol, 292, F1657 (2007) ).
15. 15. Pan, BT, Teng, K., Wu, C., Adam, M., and Johnstone, RM, Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes, J Cell Biol, 101, 942 (1985). ).
16. 16. Wolfers, J., Lozier, A., Raposo, G., Regnault, A., Thery, C., Masurier, C., Flament, C., Pouzieux, S., Faure, F., Tursz, T., Angevin, E., Amigorena, S., and Zitvogel, L., Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming, Nat Med, 7, 297 (2001). ).
17. 17. Raposo, G., Nijman, HW, Stoorvogel, W., Liejendekker, R., Harding, CV, Melief, CJ, and Geuze, HJ, B secret antigen-presenting vesicles lymphocytes, J Exp Med, 183, 1161 (1996) ).
18. 18. Thery, C., Amigorena, S., Raposo, G., and Clayton, A., Isolation and characterization of exosomes from cell culture supernatants and biological fluids, Curr Biol Biol Protocols, Chapter 3, Unit 3 22 (2006) ).
19. 19. Rubartelli, A., Cozzolino, F., Talio, M., and Sitia, R., A novel secretory pathway for interleukin-1 beta, a protein lacking signal sequence, EMBO J, 9, 1503 (1990). ).
20. 20. Frank, R., and Döring, R., Simultaneous Multiple Peptide Synthesis under Continuous Flow Conditions on Cellulose Papers Discs as Segmental Solid Synthesis, Tetrahedron, 44, 6031 (1988). ).
21. 21. Schostak, M., Schwall, GP, Poznanovic, S., Groebe, K., Muller, M., Messinger, D., Miller, K., Krause, H., Pelzer, A., Horninger, W., Klocker , H., Hennenlotter, J., Feyerabend, S., Stenzl, A., and Schrattenholz, A., Annexin A3 in urine: a highly specific noninvasive marker for prostate cancer early detection, J Urol, 181, 343 (2009). ).

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Claims (26)

1. A process for in vitro diagnosis of a prostate cancer, according to which a urine sample to be analysed is contacted with two antibodies, a capture antibody and a detection antibody, one of the two antibodies is directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, and the other of the two antibodies is directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2.
2. The process according to claim 1, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 17 consecutive amino acids of SEQ ID NO: 1.
3. The process according to claim 1, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against a polypeptide included in SEQ ID NO: 1, the amino acid sequence of which is selected from the following sequences:

   SNAQRQLIVKEYQAAYG (SEQ ID NO: 10),

   LIVKEYQAAYG (SEQ ID NO: 11)

   IVKEYQAAYGKE (SEQ ID NO: 12),

   KEYQAAYG (SEQ ID NO: 13),

   DLSGHFEHL (SEQ ID NO: 14),

   LSGHFEH (SEQ ID NO: 15),

   and

   KEYQAAYGKELKDDLKG (SEQ ID NO: 22), provided that the amino acid sequence SEQ ID NO: 22 is fused on the N-terminus side to a sequence of at least 30 amino acids.
4. The process according to claim 1, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 50 consecutive amino acids of SEQ ID NO: 2.
5. The process according to claim 4, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 45 consecutive amino acids of SEQ ID NO: 2.
6. The process according to claim 4, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope which is included in an amino acid sequence corresponding to the amino acid sequence starting at residue 3 and ending at residue 49 of SEQ ID NO: 2.
7. The process according to claim 4, 5 or 6, in which the epitope comprises in position 6 of SEQ ID NO: 2 a Lys residue.
8. The process according to claim 4, 5 or 6, in which the epitope comprises in position 6 of SEQ ID NO: 2 a Lys residue and in position 49 of SEQ ID NO: 2 an Asp residue.
9. The process according to claim 8, in which the epitope comprises in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue, and in position 9 of SEQ ID NO: 2 a Gly residue.
10. The process according to claim 4, in which the epitope comprises in position 3 of SEQ ID NO: 2 an Arg residue, in position 6 of SEQ ID NO: 2 a Lys residue, in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue, in position 9 of SEQ ID NO: 2 a Gly residue, and in position 49 of SEQ ID NO: 2 an Asp residue.
11. The process according to any one of the preceding claims, in which the antibody directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, is the capture antibody and the antibody directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2, is the detection antibody.
12. The process according to claim 1, in which the capture antibody and detection antibody are antibodies which exhibit a high affinity, with an affinity constant of at least 10^-9.
13. The process according to claim 1, in which the capture antibody and detection antibody are antibodies which exhibit a low dissociation constant of less than 2 x 10^-3 s^-1.
14. An immunoassay kit for in vitro diagnosis of a prostate cancer in a urine sample to be analysed, comprising two antibodies, a capture antibody and a detection antibody, one of the two antibodies being directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, and the other of the two antibodies being directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2.
15. A kit according to claim 14, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 17 consecutive amino acids of SEQ ID NO: 1.
16. A kit according to claim 14, in which the antibody directed against the first repeat domain of native human Annexin A3 is chosen from the antibodies directed against a polypeptide included in SEQ ID NO: 1, the amino acid sequence of which is selected from the following sequences:

    SNAQRQLIVKEYQAAYG (SEQ ID NO: 10),

    LIVKEYQAAYG (SEQ ID NO: 11)

    IVKEYQAAYGKE (SEQ ID NO: 12),

    KEYQAAYG (SEQ ID NO: 13),

    DLSGHFEHL (SEQ ID NO: 14),

    LSGHFEH (SEQ ID NO: 15),

    and

    KEYQAAYGKELKDDLKG (SEQ ID NO: 22), provided that the amino acid sequence SEQ ID NO: 22 is fused on the N-terminus side to a sequence of at least 30 amino acids.
17. A kit according to claim 14, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 50 consecutive amino acids of SEQ ID NO: 2.
18. A kit according to claim 17, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, the amino acid sequence of which comprises at least 7 consecutive amino acids, and no more than 45 consecutive amino acids of SEQ ID NO: 2.
19. A kit according to claim 17, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope which is included in an amino acid sequence corresponding to the amino acid sequence starting at residue 3 and ending at residue 49 of SEQ ID NO: 2.
20. A kit according to claim 17, 18 or 19, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 6 of SEQ ID NO: 2 a Lys residue.
21. A kit according to claim 17, 18 or 19, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 6 of SEQ ID NO: 2 a Lys residue and in position 49 of SEQ ID NO: 2 an Asp residue.
22. A kit according to claim 21, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue and in position 9 of SEQ ID NO: 2 a Gly residue.
23. A kit according to claim 17, in which the antibody directed against the fourth repeat domain of native human Annexin A3 is chosen from the antibodies directed against an epitope, said epitope comprising in position 3 of SEQ ID NO: 2 an Arg residue, in position 6 of SEQ ID NO: 2 a Lys residue, in position 7 of SEQ ID NO: 2 a Gly residue, in position 8 of SEQ ID NO: 2 an Ile residue, in position 9 of SEQ ID NO: 2 a Gly residue, and in position 49 of SEQ ID NO: 2 an Asp residue.
24. A kit according to any one of claims 14 to 23, comprising the capture antibody, which is directed against the first repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 1, and the detection antibody, which is directed against the fourth repeat domain of native human Annexin A3, the sequence of which is identified as SEQ ID NO: 2.
25. A kit according to claim 14, in which the capture antibody and the detection antibody are antibodies which exhibit a high affinity, with an affinity constant of at least 10^-9.
26. A kit according to claim 14, in which the capture antibody and detection antibody are antibodies which exhibit a low dissociation constant of less than 2 x 10^-3 s^-1.

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